PROJECT SUMMARY Genome editing with site-specific nucleases has facilitated application of reverse genetic approaches to the zebrafish model. The recent advent of highly specific and active RNA-guided nucleases have reduced cost and technical hurdles for genome editing, allowing reverse genetics to be applied in nearly all zebrafish laboratories. In the majority of published applications, these tools have been used to introduce targeted insertions or deletions (indels) by virtue of DNA repair of double strand breaks via non-homologous end-joining (NHEJ) following cleavage. A more limited number of studies have utilized homology-directed repair (HDR) with a DNA donor together with a sequence-specific nuclease to introduce sequences of interest into the zebrafish genome. While researchers have had success in this regard, rates of HDR-mediated repair events are consistently much lower than indel frequencies from NHEJ. Furthermore, many sites remain recalcitrant to knock-in at acceptable rates. This issue raises a significant barrier to the efficient production of desired knock-in alleles in the zebrafish and the widespread use of this technique in the field. Therefore, additional studies are needed to improve HDR for precise genome editing in zebrafish. In the proposed studies, we will optimize conditions for targeted knock-in to allow efficient precise genome editing in the zebrafish. In Aim 1, we will expand our application of novel parameters, identified through preliminary studies showing they that drastically improve knock-in rates, to a set of 10 loci to confirm that they are generally applicable. In all cases, we will apply definitive and accurate deep sequencing-based analysis of somatic editing rates, along with assessment of germline transmission to identify optimal parameters that are generalizable across all targets. In Aim 2, we will use optimized knock-in parameters to generate a series of zebrafish bearing Biotagged endogenous loci to enable cell type- specific enhancer and transcriptome analysis. These lines, together with the optimized parameters for precise genome editing, will serve as valuable resources for the zebrafish community.